An evo-devo geek's scientific meanderings

avian digit homology

I didn’t mean to post today, but I’ve just read a new review/hypothesis paper about the identities of the stunted little things that pass for fingers in the wings of modern birds. The review part is fine, but I’m not sure I get the difference between the hypothesis Čapek et al. (2013) are proposing and the hypothesis they are trying to replace/improve.

To recap: the basic problem with bird fingers is that fossil, genetic and developmental evidence seem to say different things about them.

1. Fossils: birds pretty clearly come from dinosaurs, and the early dinosaurs we have fossils of have five fingers on their hands with the last two being reduced. Somewhat closer to birds, you get four fingers with #4 vestigial. And the most bird-like theropods have only three fingers, which look most like digits 1, 2 and 3 of your ordinary archosaur. (Although Limusaurus messes with this scheme a bit.)

2. Embryology: in developing limb buds, digits start out as little condensations of tissue, which develop into bits of cartilage and then finger bones. Wing buds develop a short-lived condensation in front of the first digit that actually forms, and another one behind the last “surviving” digit. Taking this at face value, then, the fingers are equivalent to digits 2, 3 and 4.

3. Genetics: In five-fingered limbs, each digit has a characteristic identity in terms of the genes expressed during its formation. The first finger of birds is most like an ordinary thumb, both when you focus on individual genes like members of the HoxD cluster and when you take the entire transcriptome. However, the other two digits have ambiguous transcriptomic identities. That is, bird wings have digit 1 and two weirdos.

Add to this the fact that in other cases of digit loss, number one is normally the first to go and number four stubbornly sticks around to the end, and you can see the headache birds have caused.

So those are the basic facts. The “old” hypothesis that causes the first part of my confusion is called the frame shift hypothesis, which suggests that the ancestors of birds did indeed lose digit 1, as in the digit that came from condensation 1 – but the next three digits adopted the identities of 1-2-3 rather than 2-3-4. (This idea, IMO, can easily leave room for mixed identities – just make it a partial frame shift.)

Čapek et al.’s new one, which they call the thumbs down hypothesis, is supposedly different from this. This is how the paper states the difference:

The FSH postulates an evolutionary event in which a dissociation occurs between the developmental formation of repeated elements (digits) and their subsequent individualization.

versus

According to the TDH no change of identity of a homeotic nature occurs, but only the phenotypic realization of the developmental process is altered due to redirected growth induced by altered tissue topology. Digit identity stays the same. Also the TDH assumes that the patterning of the limb bud, by which the digit primordia are laid down, and their developmental realization, are different developmental modules in the first place.

(Before this, they spent quite a lot of words explaining how the loss of the original thumb could trigger developmental changes that make digit 2 more thumb-like.)

I…. struggle to see the difference. If you’ve (1) moved a structure to a different position, (2) subjected it to the influence of different genes, (3) and turned its morphology into that of another structure, how exactly is that not a change in identity?

Maybe you could say that “an evolutionary event” dissociating digit formation and identity is different from formation and identity being kind of independent from the start, but I checked Wagner and Gauthier’s (1999) original frame shift paper, and I think what they propose is closer to the second idea than the first:

Building on Tabin’s (43) insight, we suggest causal independence between the morphogenetic processes that create successive condensations in the limb bud and the ensuing developmental individualization of those repeated elements as they become the functional fingers in the mature hand, thus permitting an opportunity for some degree of independent evolutionary change.

So, a while ago, I went on at length about the confused identities of those stunted little bones in a bird’s wing that were once fingers. (Diagram of wing skeleton by L. Shyamal, Wikimedia Commons.)

For a brief recap: bird wings have (the remnants of) three digits. Based on palaeontological evidence (read: dinosaur hands), these would be digits I, II and III. But in a modern bird embryo, they develop in the places of digits II, III and IV. The II-III-IV hypothesis also seemed more consistent with the way other tetrapods lose digits: if five is too many, DI is typically the first to go.

To solve the apparent conundrum, Wagner and Gauthier (1999) proposed the developmental frame shift hypothesis, meaning that the developing fingers switch identities as they form. The paper I tried to discuss (and probably ended up garbling :-P) in my previous post concluded that the frame shift (if it can be called that) happens very early, before there is anything resembling actual digits in the wing bud.

Well, as always, there is a new twist on the story. (Chicken wings seem to be all the rage this year!) Wang et al. (2011) took a brute force approach towards pinning down those elusive digit identities. They compared all the genes that are active in each developing digit in both the wing and the foot of chicken embryos at two different time points. They used next-generation sequencing to catch mRNAs, the RNA copies of genes that cells make for use as templates for protein synthesis. This means thousands of genes identified from each sample, plus information on howmuch of each mRNA there is, plenty of material for comparison.

After a considerable amount of statisticking on that giant pile of data, it turns out that avian digit identities are… confused. When they grouped the different digits by how similar their gene expression patterns were, first digits proved starkly and uncontroversially well-behaved. DI from the foot (whose identity no one seriously disputes) and the first digit of the wing obviously belong together. Every other digit, though…

For example, the second finger is most like the third toe, but also kind of like the second toe. The third finger goes from being closest to the second toe to being most similar to the fourth toe as development progresses. And it’s apparently chock full of mRNA belonging to a gene that had never been reported to participate in limb development (never mind determining any digit identity) before.

With all that said, I wouldn’t be too quick to declare that wings are just weird. Well, they certainly are, but these data don’t necessarily tell us that they are weird in this particular respect. I’m sorely missing a comparison to an ordinary, five-fingered forelimb, for example. What if the weirdness is not an issue with birds, but with forelimbs?

I’d say it’s a fair bet that chicken wings will keep evo-devo nerds entertained for years to come…

(I think I’ll be discussing limb development and genetic evidence in evo-devo again in the near future. Well, for certain values of “near”. I’ve had that post in the pipeline for weeks, but the damned thing just doesn’t want to get finished.)

***

References:

Wagner GP and Gauthier JA (1999) 1,2,3 = 2,3,4: A solution to the problem of the homology of the digits in the avian hand. PNAS96:5111-5116

I’m not technically new to the blogging enterprise. I’ve kept a private journal-and-repository-of-ideas for years now, but whenever I felt like I had something to say that was worth sharing, I inevitably changed my mind. But, with so much wonderful, exciting stuff out there, isn’t it practically a crime for a scientist not to share?

I thought it fitting to start my science blogging career with tetrapod limbs, since they played a great part in setting me on the path that led me where I am today. If I had to define my specialisation, I would call myself an evolutionary developmental geneticist. (Add “molecular” in there somewhere to make it more accurate and more cumbersome ;)) This means investigating the genes that control the formation of body parts in various organisms, and trying to figure out what this tells us about the evolution of said body parts. One of the early signs that alerted me to the awesomeness of the field was a paper about limbs (Davis et al., 2007) – more precisely, about how the genetic program that specifies our fingers and toes is present in the fins of fish with nothing resembling digits. That is an intriguing story that I might tell another day, but today, I will discuss something else to do with limbs, apropos a study that came out recently in Science.

Birds have some of the most extremely modified forelimbs among tetrapods. If you squint at the skeleton inside the wing hard enough, you may be able to recognise the stunted remains of three digits. The homology of those three digits to the digits of more conventional forelimbs has been a conundrum ever since someone first examined the limb buds of a bird embryo.

Homology is a tricky concept. It was originally defined before the theory of evolution took off, by a guy who had a rather strange relationship with Darwin and Wallace’s theory later on. Richard Owen (see here for a biography) defined homology as:

the same organ in different animals under every variety of form and function

Later, evolutionary biology embraced the word as a neat shorthand for structures, processes, genes, even behaviours, that evolved from a common ancestor. The obvious problem is that organs and genes don’t come with labels listing what they are homologous to. Therefore, homology has to be inferred. These inferences can be based on a variety of sources such as morphological similarity, fossil evidence, embryonic origin and shared genetic programs. The more lines of evidence converge on the same conclusion, the stronger the conclusion is. But what happens when those different sources contradict?

In the case of avian digits, morphology [1], and later palaeontology, suggested that they were digits I, II and III, that is, a thumb, an index and a middle finger. While it’s a bit hard to say anything about the morphology of those fused and stunted bones in a modern wing with a straight face, the fossil evidence is pretty unambiguous. As you go from the earliest dinosaurs to birds, you can clearly follow the loss of digit V first, followed by digit IV. The remaining three digits are quite clearly I-II-III in animals such as Archaeopteryx.

A comparison of the metacarpals (palms) of dinosaurs. Left to right, in order of increasing relatedness to birds: Herrerasaurus (note the vestigial fifth metacarpal (labelled "V")), Coelophysis (MCV gone), Allosaurus (MC4 gone), Deinonychus, Archaeopteryx, and Nothura (a modern tinamou). Image from Wagner and Gauthier (1999)

However, embryos seemed to tell a completely different story. During development, the skeleton of a limb forms from little condensations of tissue inside the limb bud (which, at that point, looks more like a weird-shaped sausage than a limb). These condensations are first cartilaginous, later laying down bone. Around the forming digits, muscles and connective tissues organise, and finally, the padding between the digits dies away, transforming the paddle-like limb bud into a hand or foot [2]. The forelimbs of birds make four condensations – more than needed to form their three digits, but fewer than five, making it difficult to tell exactly which ones remain.

Nevertheless, regularities observed in limb development gave scientists clues. The condensations that turn into digits in tetrapods don’t all form at the same time – in fact, they form in a stereotypical order in which CIV forms first and CI last (Burke and Feduccia, 1997). Condensation (and digit) IV is considered part of the main axis of the limb (the so-called metapterygial axis), and its presence is thought to be essential for the other condensations to form. You cannot lose CIV without losing all the rest, so conventional wisdom went. That means that birds must have a CIV. And indeed, the third digit of a wing comes from something that looks suspiciously like a CIV, with two other condensations appearing in front of it and one behind. Thus, embryology would have us think that the digits are II, III and IV.

Diagram comparing the development of the forelimb skeleton in alligators (top) and ostriches (bottom). Image from Wagner and Gauthier (1999).

This presents a dilemma that is a common one in evo-devo. Which kind of evidence do you put more stock in? Is there a way to resolve the conflict, or does it spell doom for the hypothesis that birds evolved from dinosaurs (and then, what’s up with the four- and three-fingered dinosaurs that obviously aren’t birds?)? What’s going on here?

An important paper (Wagner and Gauthier, 1999) over a decade ago suggested a possible resolution called the developmental frame shift hypothesis. They proposed that birds do in fact form condensations II-IV – but then something strange happens. The genetic program that specifies digit identity is not switched on until all pre-digital condensations have formed. Thus, a condensation can become any digit, depending on which genes are expressed at its location. So, Wagner and Gauthier said, at some point in their history, dinosaurs shifted the “normal” forelimb gene expression patterns backwards, so CII now ended up in the genetic environment of DI, and thus formed a DI. That is, the fingers of birds are homologous to different digits of the basic pentadactyl limb at different levels.

Over the years since, strong genetic support has been gathered for this idea. The first digit of the hand of a bird does, indeed, form in an area that bears the genetic hallmarks of DI in more typical tetrapods, including the closest living relatives of birds (Vargas et al., 2008). So, the frameshift hypothesis of Wagner and Gauthier seems to stand on pretty solid legs.

A recent study had a new look at wing development, confirming the identity of wing digits as I-III. Tamura and others (2011) followed the origin of digits at the cellular level. An early limb bud, before it has much obvious internal structure, already contains what developmental biologists call organisers or signalling centres. At the tip of the bud, a narrow ridge (the so-called apical ectodermal ridge, or AER) gives off chemical signals that direct the correct outgrowth of the limb. At the posterior, tailward side of the bud, there is an area called the Zone of Polarising Activity, which defines the anterior-posterior polarity of the limb: the side of the ZPA is the pinky side, and where everything else develops is determined by the levels of morphogens – most famously the protein Sonic hedgehog – that diffuse from the ZPA.

In ordinary, five-fingered tetrapods, the ZPA contributes tissue to the fifth and fourth condensations (and digits). The first thing Tamura and colleagues did, then, was to transplant ZPAs between the fore- and hindlimb buds of chick embryos (remember, chicken legs are four-toed). When you add a ZPA on the wrong side of a limb bud, you get a mirror image duplication of the limb. (How much is duplicated depends on the age of the transplant and the recipient.) In this study, the researchers found that a hindlimb ZPA of the right age grafted onto a forelimb bud often led to the formation of hindlimb digits in the forelimb. The converse was not true: the forelimb ZPA was almost invariably unable to form forelimb digits, though it was able to induce the doubling of the foot. Thus, it seems cells in the forelimb ZPA aren’t good at making digits on their own, only at directing other tissues to form digits.

The second experiment involved tracing cell lineages with a clever method that’s widely used in similar experiments due to its relative simplicity. They stained cells with a dye that sticks to the cell membrane. This dye is inherited by the descendants of the mother cell: when it divides, the daughter cells will split the original membrane – and the dye – between them. So, if the ZPA contributes to the last digit of the wing, we should see staining in the digit if we’d dyed the ZPA before digits begin to condense. In the foot, that’s exactly what happens. Not so in the wing: the last digit forms outside the ZPA, with no contribution from the labelled cells.

And lastly, the researchers went further back in time, using the same cell labelling method to trace the fate of different parts of the limb bud from an even earlier stage. What they found is that the region that gives rise to the third finger does lie inside the ZPA at this point – but it leaves the zone very soon after, unlike the progenitor of the fourth toe. Thus, well before any condensation is apparent, this bit of tissue is different from conventional fourth digits.

Interestingly, what this study identifies as a digit III based on its origin appears to lie on the metapterygial axis – a place thought to be the privilege of fourth digits. What’s more, removing the posterior portion of the limb bud relocates the second digit to the main axis. Thus, it appears that limb development is a lot more flexible than Burke and Feduccia had concluded over a decade earlier.

So, in a sense, Wagner and Gauthier were wrong – but in a different sense, they were more right than they thought. It appears that the frame shift they hypothesised doesn’t happen after condensations appear: it happens well before. What was previously identified as “CIV” already bears characteristics of CIII when the first lumps of cartilage begin to form.

In a way, the story of avian digits is a beautiful illustration of the scientific process. From a controversy sparked by a seemingly insurmountable contradiction, we have moved to a synthesis that accounts for all available evidence. Scientists did not dismiss the contradiction, they worked to make sense of it. They called on new lines of evidence to resolve what the old evidence could not. Some ideas proposed in the process – such as the rigidity of the digit developmental program – turned out largely wrong. Others – the original frame shift hypothesis – still seem somewhat wrong, but their essence carried over into the newest picture. It’s entirely possible that this isn’t the last word on bird fingers either. But at this point, I am reminded of Asimov’s essay The Relativity of Wrong (Asimov, 1989). Whatever the future brings, it’s a good bet that we’re not nearly as “wrong” as we were before.

– – –

[1] Comparing ordinary, five-fingered forelimbs, some characteristics are pretty consistent across the different groups of tetrapods. Most importantly, thumbs have only two phalanges, fewer than all the other digits.

[2] This isn’t universally the case. When the interdigital tissue doesn’t disappear, you get webbed feet.

– – –

References:

Asimov I (1989) The Relativity of Wrong. The Skeptical Inquirer14:35-44

Burke AC and Feduccia A (1997) Developmental patterns and the identification of homologies in the avian hand. Science278:666-668

Davis MC, Dahn RD and Shubin NH (2007) An autopodial-like pattern of Hox expression in the fins of a basal actinopterygian fish. Nature447:473-476